Metastasis is the leading cause of cancer death. Once cancer has spread to and beyond regional lymph nodes, complete eradication of the tumor by surgical intervention is nearly impossible (S. A. Rosenberg, Cancer: Principles and Practice of Oncology, V. T Devita, S. Hellman and S. A. Rosenberg, Eds. (Lippincott-Raven, Philadelphia, 1997), p. 295). Instead, the whole body must be treated with radiation or chemotherapy, which are notoriously toxic to normal cells and tissues (V. T. Devita, Cancer: Principles and Practice of Oncology, V. T. Devita, S. Hellman and S. A. Rosenberg, Eds. (Lippincott-Raven, Philadelphia, 1997), p. 333). These facts underscore the need to identify the fundamental causes of metastasis and to translate this information into more effective and less toxic therapies. In spite of recent advances in studies of the basic mechanisms of metastasis, relatively little of this progress has translated into therapeutic approaches that minimize damage to normal tissues.
Metastasis
Recent investigation reveals that micrometastatic tumors can disseminate throughout the body long before cancer is first detected (D. E. Henson, et al, Current Opinion in Oncology 11:419, 1999) (I. J. Fidler, Cancer: Principles and Practice of Oncology, V. T. Devita, S. Hellman and S. A. Rosenberg, Eds. (Lippincott-Raven, Philadelphia, 1997), p. 135). This fact underscores the need to identify the causes of micrometastasis, with the ultimate goal of translating this information into new therapies. Growth and survival of benign and metastatic cells are differentially regulated by extracellular matrix (ECM) adhesion (J. A. Lawrence, et al., World Journal of Urology 14:124, 1996). Specifically, the growth and survival of benign epithelial cells requires cell attachment the basal laming. At the biochemical level, ECM anchorage generates signals that are necessary for growth and survival (E. Ruoslahti, Advances in Cancer Research 76:1, 1999). Consequently, the growth and survival of benign cells are compromised when they detach from the basement membrane or are transplanted into a foreign microenvironment (S. M. Frisch, et al., Current Opinion in Cell Biology 9:701, 1997). In contrast, metastatic cells grow and survive independent of changes in the local microenvironment (J. A. Lawrence et al., 1996). Unfortunately, most studies of metastatic cell growth and survival rely upon monolayer cell culture and thus fail to exploit important differences between normal and metastatic cells. Indeed, increasing evidence reveals that monolayer culture does not reliably model tumor cell behavior in vivo (J. A. Lawrence et al., World Journal of Urology 14:124 (1996) (V. M. Weaver, et al., Semin Cancer Biol 6:175, 1995).
Cancer Cell Signaling
Cancer is a disease of aberrant signal transduction. Aberrant cell signaling overrides anchorage-dependent constraints on cell growth and survival (J. S. Rhim, et al., Critical Reviews in Oncogenesis 8:305, 1997; R. Patarca, Critical Reviews in Oncogenesis 7:343, 1996; R. K. Malik, et al., Biochimica et Biophysica Acta 1287:73, 1996); (W. G. Cance, et al., Breast Cancer Res Treat 35:105, 1995). Tyrosine kinase activity is induced by ECM anchorage and indeed, the expression or function of tyrosine kinases is usually increased in malignant cells (J. S. Rhim, et al., Critical Reviews in Oncogenesis 8:305, 1997; W. G. Cance, et al., Breast Cancer Res Treat 35:105, 1995; T. Hunter; Cell 88:333, 1997). Based on evidence that tyrosine kinase activity is necessary for malignant cell growth, tyrosine kinase have been targeted with new therapeutics (A. Levitzki, et al., Science 267:1782, 1995; B. S. Kondapaka, et al., Molecular & Cellular Endocrinology 117:53, 1996; D. W. Fry, et al., Current Opinion in Biotechnology 6: 662, 1995). Unfortunately, obstacles associated with specific targeting to tumor cells often limits the application of these drugs. In particular, tyrosine kinase activity is often vital for the function and survival of benign tissues (A. Levitzki, et al., Science 267:1782, 1995). To minimize collateral toxicity, it is critical to identify and then target tyrosine kinases that are selectively overexpressed in tumor cells.
New technologies to identify tyrosine kinases that are overexpressed or functionally altered in metastatic carcinoma cells are available (M. S. Kinch, et al., Hybridoma 17:227, 1998). Strategies were used to generate monoclonal antibodies against tyrosine kinases and their substrates in avian fibroblasts (S. B. Kanner, et al., Proc Nad Acad Sci USA 87:3328, 1990; J. R. J. Glenney, et al., J Cell Biol 108:2401, 1989). A small number of antigens were identified in these earlier studies. RIMMS immunization strategy was used to increase the breadth and sensitivity of monoclonal antibody production to generate monoclonal antibodies against tyrosine kinases in human breast carcinoma cells (Kinch et al., 1997, which is incorporated herein by reference in its entirety for the RIMMS method). RIMMS involves repetitive immunizations with low-dose antigen into multiple sites, all of which are proximal to draining lymph nodes. An abbreviated course of immunization promoted affinity maturation while minimizing immunodominance. RIMMS allowed us to isolate numerous monoclonal antibodies that recognize tyrosine kinases and their substrates in metastatic cells. The antibodies were screened for antigens that were differentially expressed in non-transformed versus metastatic epithelial cells. Two antibodies, D7 and B2D6, were identified that recognized an antigen that was grossly overexpressed and functionally altered on breast and prostate cancer cells (N. D. Zantek, et al, Cell Growth & Differentiation 10:629, 1999; J. Walker-Daniels, et al, Prostate 41: 275, 1999). The antigen was identified with a phage-expression library as the EphA2 tyrosine kinase.
EphA2
EphA2 is a 130 kDa receptor tyrosine kinase that is expressed in adult epithelia, where it is found at low levels and is enriched within sites of cell-cell adhesion (N. D. Zantek, et al, Cell Growth & Differentiation 10:629, 1999; R. A. Lindberg, et al., Molecular & Cellular Biology 10: 6316, 1990). This subcellular localization is important because EphA2 binds ligands (known as Ephrins A1 to A5) that are anchored to the cell membrane (J. G. Eph Nomenclature Committee (Flanagan, N. W. et al., Cell 90, 403 (1997; N. W. Gale, et al., Cell & Tissue Research 290: 227, 1997). The Ephrin ligands, in turn, can bind any of 8 different EphA family kinases. The primary consequence of ligand binding is EphA2 autophosphorylation (R. A. Lindberg, et al., 1990). However, unlike other receptor tyrosine kinases, EphA2 retains enzymatic activity in the absence of ligand binding or phosphotyrosine content (N. D. Zantek, et al, 1999). Consequently, we define ligand-mediated “activation” as increased EphA2 phosphotyrosine content (N. D. Zantek, et al., 1999). Because most Eph kinase family members are expressed in the embryonic nervous system (E. B. Pasquale, Current Opinion in Cell Biology 9:608, 1997), investigators studying Eph kinases have largely overlooked EphA2, which is primarily on adult epithelial cells (R. A. Lindberg, et al., 1990). Studies of EphA2 have been further limited by a lack of reagents and model systems.